Abstract

MicroRNAs are pervasive in both plants and animals, but many aspects of their biogenesis,
function and evolution differ. We reveal how these differences contribute to characteristic
features of microRNA evolution in the two kingdoms.

Review

The first microRNAs (miRNAs) to be discovered, lin-4 and let-7, were found to be regulators
of Caenorhabditis elegans development [1-3], and they established a paradigm for eukaryotic gene regulation in which short hairpins
generate RNAs of approximately 22 nucleotides (nt) that repress specific target mRNAs.
miRNAs have proved to be pervasive in both animals [4-6] and plants [7,8], acting as sequence-specific guides for target recognition [9,10]. Several thousand miRNAs have now been found in dozens of plants and animals [11]. Moreover, the biogenesis and activity of miRNAs are strongly related to those of
small interfering RNAs (siRNAs) that mediate RNA interference, another ancient mechanism
for post-transcriptional gene silencing [12].

Although miRNAs mediate diverse aspects of development and physiology in both plants
and animals [13,14], there are substantial differences between them. For example, the loci that produce
miRNAs have distinct genomic arrangements in each kingdom. Furthermore, miRNAs are
excised from precursor transcripts by different pathways in the two kingdoms, and
in different subcellular compartments. Once made, plant and animal miRNAs have vastly
different suites of direct targets; the number of direct targets of a given animal
miRNA generally exceeds that of a given plant miRNA by at least an order of magnitude
[15]. Herein, we focus on how these differences contribute to, and are the result of,
distinct evolutionary characteristics of miRNAs in the two kingdoms. We also highlight
many commonalities between the respective systems that may reflect a shared evolutionary
heritage or convergent strategies for handling and metabolizing double-stranded RNAs.

Distinct characteristics of miRNA pathways in plants and animals

What is a miRNA? Answering this question is not a simple task, as no single definition
clearly and specifically encompasses all miRNAs. Although practical guides for miRNA
annotation in plants and animals exist [16,17], not all loci reported in the miRBase registry [18] have been annotated to the same degree of confidence. In general, miRNAs are the
products of inverted repeat transcripts that are precisely cleaved by RNase III enzyme(s)
in the Dicer and/or Drosha protein families to yield small RNAs of approximately 21
to 24 nucleotides that guide Argonaute (AGO) proteins to complementary targets. Analogous,
but distinct, core pathways govern the biogenesis of most miRNAs in plants and animals.
Although we focus on these canonical miRNA pathways, a plethora of alternative pathways
exist. Indeed, the diversity and flexibility of miRNA biogenesis pathways, in concert
with related mechanisms that generate siRNAs, have made a significant contribution
to miRNA evolution. In addition, while a hallmark of most studied miRNAs is the precise
manner in which they are excised from precursor hairpins, there are examples of imprecisely
cleaved miRNAs. As we shall see, this phenomenon might have implications for miRNA
evolution, but it also poses challenges for the accurate distinction of bona fide miRNAs from fortuitous hairpins associated with short RNAs not generated by a specific
biogenesis machinery.

Canonical miRNA biogenesis in plants versus animals

The biogenesis of plant miRNAs has been documented most thoroughly in Arabidopsis thaliana (Figure 1a). Primary miRNA (pri-miRNA) transcripts are products of RNA polymerase II that contain
a hairpin RNA secondary structure [19]. The length of plant pri-miRNA hairpins is heterogeneous, ranging from approximately
70 to many hundreds of bases. Of the four Dicer-like enzymes in Arabidopsis, Dicer-like 1 (DCL1) is responsible for the bulk of miRNA biogenesis [20]. DCL1 usually cleaves from the base of the pri-miRNA hairpin to yield a precursor-miRNA
(pre-miRNA) hairpin, and cleaves again to release a miRNA/miRNA* duplex [21], although 'loop-first' processing, where the first DCL1-catalyzed cut occurs proximal
to the loop, can also occur [22,23]. Most plant pri-miRNA hairpins produce a single miRNA/miRNA* duplex, but some loci,
including MIR159 and MIR319, consistently produce multiple duplexes [22-24].

Figure 1.Major biogenesis pathways of small RNAs from inverted repeat transcripts in plants
and animals. (a) In plants, canonical microRNAs (miRNAs) are produced by the nuclear RNase III Dicer-like1
(DCL1), which cuts from the base of the hairpin towards the loop; a subset of plant
miRNAs are processed from the loop towards the hairpin. One miRNA/miRNA* duplex is
shown, but there can be several such duplexes depending on the length of the stem.
These are transported from the nucleus via HASTY, an Exportin5 (Exp5) homolog, for
loading into an Argonaute (AGO) complex. The main miRNA effector in plants is AGO1,
and to a lesser extent AGO10 and other AGOs; AGO7 carries the exceptional miRNA miR390.
Long well-paired hairpins (proto-miR/inverted repeat (IR) transcripts) can be processed
by a diversity of Dicers to generate either miRNAs or small interfering RNAs (siRNAs).
The subcellular location for dicing by DCL2 and DCL4, and subsequent AGO loading of
the resulting siRNAs, is not yet clear. nt, nucleotide. (b) In animals, canonical miRNAs are processed by the nuclear RNase III enzyme Drosha.
The precursor miRNA (pre-miRNA) hairpin is exported to the cytoplasm by Exp5 to generate
a single miRNA/miRNA* duplex, which is loaded into a miRNA class AGO protein (Drosophila dAGO1, Caenorhabditis elegans ALG1/2, or vertebrate Ago1 to Ago4). There are Drosha-independent non-canonical pathways,
including the mirtron pathway where intron splicing and lariat debranching generate
pre-miRNA hairpins. Also, vertebrate miR-451 is matured by a Dicer-independent route.
Here, Drosha cleavage generates a short hairpin that is loaded into the 'Slicer' Ago2,
which cleaves its 3' arm; this is resected to yield the mature miRNA. Unlike other
vertebrate miRNAs, miR-451 can only be matured in Ago2. Finally, in the Drosophila hairpin RNA pathway, long inverted repeats are processed by the endogenous siRNA pathway,
being cleaved by d-Dicer2 to generate siRNAs that load dAGO2. Many Drosophila miRNA* species are also preferentially sorted into dAGO2 (dashed arrow).

Plant miRNA/miRNA* biogenesis is completed within the nucleus [25] in specialized subnuclear regions termed D-bodies [26,27]. Several accessory factors also contribute to the efficiency and fidelity of miRNA/miRNA*
excision in plants (for a recent review, see [14]). The 3'-most nucleotides of the initial miRNA/miRNA* duplex are then 2'-O-methylated by the nuclear HEN1 protein [28]; this modification prevents non-templated 3'-polymerization that accelerates miRNA
turnover [29]. HASTY, a plant homolog of Exportin-5, is then thought to export miRNA/miRNA* duplexes
for loading into cytoplasmic AGO proteins [25], of which AGO1 is the predominant carrier of plant miRNAs. AGO1 can act as a 'slicer'
to direct the endonucleolytic cleavage of target RNAs [30,31]; most other plant AGOs are also likely to possess slicing capabilities [32].

The collected studies from Drosophila, C. elegans and mammalian cells [12] indicate a conserved animal mechanism that is analogous to, but distinct from, plant
miRNA biogenesis (Figure 1b). Most animal miRNAs are transcribed by RNA polymerase II, although a subset of animal
miRNAs are products of RNA polymerase III [33]. The major difference compared with plants is the segregated cleavage of miRNA precursors
by nuclear and cytoplasmic RNase III enzymes. All animals use the Drosha RNase III
enzyme, which partners with the double-stranded RNA-binding domain protein DGCR8 (known
as Pasha in invertebrates), to liberate pre-miRNA hairpins from pri-miRNA transcripts.
The lengths of pre-miRNAs are more consistent in animals than in plants, with most
in the 55- to 70-nucleotide range; however, select Drosophila pre-miRNAs can approach 200 nucleotides [34].

Following nuclear export of pre-miRNAs by Exportin-5, they are cleaved into miRNA/miRNA*
duplexes by cytoplasmic Dicer (a single enzyme in C. elegans and vertebrates, and Dicer-1 in Drosophila). These are loaded into miRNA effector Argonautes (Drosophila dAGO1, C. elegans ALG1/2, and vertebrate Ago1 to Ago4). Of the mammalian Ago proteins, only Ago2 has
Slicer activity [35,36]; Drosophila dAGO1 has Slicer activity, but appears to have poorer turnover than its paralog dAGO2,
the major carrier of endogenous siRNAs (endo-siRNAs) [37,38]. Curiously, while plant miRNAs are universally methylated at their 3' ends by HEN1,
most products of animal miRNA genes are not. An exception regards Drosophila miRNA* strands, which are preferentially loaded into dAGO2 (Figure 1b). All dAGO2 cargoes, including miRNA* strands, endo-siRNAs and exogenous siRNAs from
viruses or artificial dsRNA, are methylated by HEN1 as single-stranded species [39-42]. In addition to this core machinery, several accessory factors influence the biogenesis
efficiency, fidelity and sorting of animal miRNAs [43]. Notably, a growing number of these factors act in cell-specific or state-specific
manners to regulate miRNA production or activity, indicating that neither process
is constitutive.

Genomic arrangement of plant and animal miRNA genes

In plants, most miRNA-encoding loci comprise independent, non-protein-coding transcription
units. Among the rare intronic plant miRNAs is one present within an intron of DCL1 orthologs; this miRNA might direct feedback regulation of miRNA biogenesis [44,45]. Plant miRNA hairpins sometimes occur in genomic clusters, strongly suggesting expression
of multiple hairpins from a single pri-miRNA. Around one-fifth of annotated miRNAs
in Arabidopsis, rice and poplar occur in tandem clusters at distances less than 10 kb [46]. Most clusters in these species (61% to 90%) contain hairpins encoding identical
mature miRNAs, suggesting that they were the result of local tandem duplications and
serve to increase the dosage of a particular miRNA from a single promoter [46]. The minority of plant miRNA clusters that produce more than one mature miRNA family
nearly all encode mature species that are not conserved outside of the genus within
which they were first described.

The genomic patterns of animal miRNA genes are significantly different from those
of plants. Although many derive from stand-alone non-protein-coding loci, approximately
30% are located on the sense strands of introns [47]. There are only a few cases of miRNAs transcribed antisense to introns, suggesting
some evolutionary benefit to sense intronic location. The simplest notion is that
this arrangement permits miRNAs to take advantage of cis-regulatory elements that direct the expression of the host mRNA; however, intronic
miRNAs can also be controlled by independent regulatory elements [48-50]. Unlike in plants, there are also occasional examples of miRNA biogenesis from exons
of animal protein-coding genes, including UTRs and coding sequences (CDSs) [51,52].

Clustering of miRNA genes is more common in animals than in plants: up to 40% of miRNAs
in nematodes, flies and mammals are clustered in their respective genomes. Curiously,
while there are many cases of locally duplicated miRNA hairpins, it is common for
animal miRNA clusters to encode unrelated mature miRNAs. Only 5% to 20% of operons
are composed exclusively of duplicated miRNAs in these three well-studied animal clades.
Therefore, amplification of specific miRNA levels is not sufficient to explain the
composition of animal miRNA operons. Instead, these different genomic origins and
arrangements reflect distinct evolutionary styles of canonical miRNAs in plants and
animals.

Many non-canonical mechanisms convert precursor transcripts into miRNAs and/or siRNAs.
The strategies that are most relevant to miRNA evolution are pathways that metabolize
inverted repeat transcripts (for reviews, see [53,54]). In animal cells, the first major alternative to the canonical miRNA pathway came
with the recognition of mirtrons (Figure 1b), which are pre-miRNA hairpins whose ends are defined by splicing instead of Drosha
cleavage [55-57]. Following their debranching into a linear form, they are diced into conventional
miRNAs. Mirtron biogenesis has not been extensively documented in plants, but one
short hairpin intron in rice (MIR1429) generates specific miRNA/miRNA* reads indicative of mirtron processing [58].

In addition to other types of Drosha-independent miRNAs in animals [59], the conserved vertebrate miR-451 matures by a Dicer-independent mechanism [60-62]. Following Drosha cleavage, the pre-mir-451 hairpin has only 19 bp of stem, which is too short to be cleaved by Dicer. Instead,
it is loaded directly into Ago2, the sole vertebrate Argonaute-class Slicer enzyme.
Ago2 cleaves its 3' hairpin arm generating a 30-nucleotide species, whose 3' end is
resected to yield the mature miRNA of approximately 23 nucleotides (Figure 1b). The miR-451 pathway is instructive in that it does not proceed via a miRNA/miRNA*
intermediate; typical annotation strategies require such paired duplexes for confident
miRNA calls. AGO-mediated miRNA biogenesis from short hairpins has not been reported
in plants to date.

In Drosophila, artificial long inverted repeat transcripts efficiently silence homologous transcripts,
permitting transgenic RNAi [63]. These exogenous triggers prefaced the recognition of endo-siRNA substrates, including
hairpin RNAs (hpRNAs). These long structured hairpins resemble long miRNA hairpins,
in that they have internal mismatches and bulged positions; however, they are not
processed by miRNA machinery. Instead, they traverse a siRNA pathway (Figure 1b), and are cleaved by Dicer-2 to generate duplexes that are preferentially loaded
into AGO2 [64-66]. Endogenous processing of long hairpins in mammalian cells appears limited, because
extensive dsRNAs trigger the antiviral interferon response. Some hpRNA-type loci are
expressed and processed in embryonic stem cells and ovaries, indicating that this
mode of biogenesis exists in vertebrates [67-69].

In contrast to vertebrates, plants have an extensive capacity to process long inverted
repeat transcripts into small RNAs. There are currently no examples of well-conserved
hairpin small RNA loci in plants, but they might conceivably play species-specific
roles [70]. However, as in Drosophila, artificial hairpins are useful for reverse genetics in plants, indicating that perfect
inverted repeat transcripts are readily accepted by small RNA biogenesis pathways.
Interestingly, there exists a clear continuum of hairpin-derived small RNAs in plants,
ranging from canonical miRNAs (defined by the precise production of a discrete miRNA/miRNA*
duplex, or phased duplexes) to heterogeneously processed hairpins exhibiting enormous
size variation [45,71]. The more imprecisely processed plant hairpins are generally processed by one or
more of the Dicer-like enzymes DCL2, DCL3 or DCL4 (Figure 1a), which are also associated with production of dsRNA-derived siRNAs of various functions
[45,70]. This continuum highlights the subjective nature of annotating miRNAs, the hallmark
of which is the precision of their small RNA ends. It is a particular challenge to
categorize ambiguous plant inverted repeats that generate an abundance of reads, of
which only a subset conform to putative miRNA/miRNA* duplexes.

Target recognition by plant and animal miRNAs

The substantial differences between the biogenesis of animal and plant miRNAs are
also reflected in the differences in their requirements for target recognition. It
has long been known that plant miRNAs often have targets with perfect [72] or, more frequently, near-perfect [10] complementarity, facilitating relatively simple identification. Canonical plant miRNA
target sites are found in 5' UTRs, ORFs and 3' UTRs, as well as within non-protein-coding
transcripts, suggesting that all RNA contexts are equally amenable to miRNA-directed
regulation in plants. Many of these plant miRNA targets succumb to AGO-catalyzed cleavage
when they encounter a cognate miRNA; the characteristic remnants of these cleavage
reactions enable molecular confirmation of plant miRNA target predictions in vivo [72-74]. However, not all plant miRNA-target interactions lead to AGO-catalyzed slicing.
Some plant miRNA targets have conserved central mismatches embedded within perfectly
base-paired regions at the 5' and 3' ends that allow AGO/miRNA binding but prevent
slicing [75,76], whereas others seem to be refractory to target cleavage despite extensive complementarity
[77]. In addition, even for plant miRNA targets that are sliced, slicing is often not
the sole mechanism by which miRNAs repress target gene expression in plants: several
experiments, involving multiple plant miRNA families, have demonstrated a pervasive
contribution of translational repression to plant miRNA function [77-79].

Whether operating via slicing and/or translational repression, though, most evidence
indicates that plant miRNAs require extensive pairing to their targets. By contrast,
it is rare for animal miRNAs to identify targets with 'plant-like' complementarity.
The initial target sites identified for the founding miRNA lin-4, within the 3' UTR
of C. elegans lin-14, exhibited only partial complementarity [1,2]. Subsequent studies in the Drosophila system elucidated arrays of approximately seven-nucleotide conserved 3' UTR motifs
termed Brd boxes, GY boxes and K boxes, which mediate critical post-transcriptional
repression of Notch target genes during sensory bristle and eye development [80-82]. These motifs proved to represent binding sites for most of the initially described
Drosophila miRNAs [5], and defined their capacity to identify targets via complementarity to their 5' ends,
preferentially at nucleotides 2 to 8 [9]. Extensive computational and experimental studies verified this as the major mode
of miRNA target recognition in animals, with Watson-Crick pairing of positions 2 to
8 of the miRNA referred to as 'seed-pairing' [83,84]. Additional features, such as an adenosine following the seed match, the location
within the 3' UTR, proximity to other miRNA-binding sites, and the degree of local
secondary structure, also influence target site activity [15].

Although there is also clear evidence for evolutionary selection of animal miRNA-binding
sites in coding regions or even 5' UTR sites [85,86], most of the well-studied target sites in this kingdom occur in 3' UTRs. The efficacy
of CDS or 5' UTR sites appears to be hampered owing to competition with ribosomes
[87]. Since artificial siRNAs that guide target slicing operate efficiently via coding
sites in animal transcripts, similar to plant miRNAs, the paucity of CDS targeting
is consistent with the view that relatively little miRNA target regulation in animals
is mediated by AGO-catalyzed slicing; indeed, efforts to identify sliced remnants
of AGO-catalyzed cleavage in mammalian samples yield very few targets [88,89]. The molecular mechanism(s) of miRNA targeting remain under investigation, and many
have been proposed [90,91]. Although much attention has been focused on translational inhibition mechanisms,
there is also evidence that bulk regulatory properties of animal miRNAs can be explained
by mRNA degradation, possibly through induction of deadenylation [80,81,92,93].

In vertebrates, about 30% of transcripts contain probable miRNA-binding sites that
have been conserved between mammals and chicken [94,95], and a comparable breadth of targeting has been detected in invertebrates [34,96,97]. Genome-wide transcriptome [92,98] and proteome [99,100] studies provide experimental support for the breadth of miRNA targeting in animal
cells, and further indicate that many functional miRNA:target interactions are not
well conserved. Moreover, at least some functional animal miRNA binding sites lack
seed-pairing [2,3,101]. miRNA targeting in animals has broad potential to be combinatorial, since individual
targets often bear conserved target sites for different miRNAs [94,95]. The scope of miRNA targeting appears to be drastically different in plants. Less
than 1% of the transcripts in Arabidopsis are known or predicted miRNA targets [20,74] and there appears to be little, if any, combinatorial control. Almost all known plant
miRNA targets have a single target site and are regulated by just one miRNA. These
genome-wide principles may support the notion that animal miRNAs generally cast a
wide net of mostly subtle regulatory effects across the transcriptome, while plant
miRNAs have more focused and stronger regulatory effects on a relative handful of
key targets. Of course, these generalizations should not be over-interpreted. Although
quantitatively mild regulation can be of substantial importance to normal development
or physiology, the loss of potent regulatory interactions is sometimes of surprisingly
minimal phenotypic consequence [13].

Overall, the pairing requirements and target breadth of animal and plant miRNAs are
clearly distinct, and this might be related to differences in their evolutionary emergence.
Nevertheless, there is reason to believe that evolution has acted upon a backdrop
of shared ancestral mechanisms. A recent study showed that Drosophila endo-siRNAs loaded in AGO2 require their 2'-O-methylation to remain stable in the presence of highly complementary targets [102]. Reciprocally, instability of Drosophila miRNAs loaded in AGO1 was induced by providing them an artificial, perfectly complementary
target, and similar findings applied to mammalian miRNAs. It was proposed that the
general rarity of highly complementary targets for animal miRNAs has permitted them
to dispense with a 3' protection pathway [102]. By contrast, the fact that most plant miRNAs do have highly complementary targets
may provide a pressure for obligate 3' methylation of plant miRNAs. Moreover, the
piwi-interacting RNA (piRNA) class of small RNAs carried by animal Piwi proteins are
also methyated by Hen1 [42,103,104], and key functions of piRNAs include the recognition of perfectly matching transposon
transcripts [105]. These data suggest that an evolutionarily ancient aspect of small regulatory RNA-mediated
regulation is sensitive to the status of target pairing.

Contrasting modes of evolutionary emergence of plant and animal miRNAs

miRNA formed from intragenomic duplication

How are new miRNAs formed? An important clue came from the observation that some recently
evolved miRNA hairpins in plants exhibit complementarity with their target mRNAs that
extends beyond the region of the mature miRNA [106]. This observation suggested an evolutionary scenario where an inverted duplication
of a gene gave rise to a 'proto-miRNA', which, when transcribed, would make a hairpin
capable of producing small RNAs with perfect complementarity to the parental transcripts
(Figure 2a). Over time, mutational drift obscures the extensive homology to the parental transcript
and refines the precision of small RNA processing, leaving just a single region (the
mature miRNA) that retains complementarity (Figure 2b). Consistent with this hypothesis, evidence for extended complementarity of plant
miRNA hairpins to target mRNAs is restricted to less-conserved (and therefore presumably
younger) loci [20,106,107].

Figure 2.Modes of microRNA emergence in plants and animals. (a) Left: intragenomic duplications of protein-coding genes (or non-coding regions) can
generate long foldbacks, which can be diced into small RNAs capable of targeting the
progenitor transcript. This phenomenon seems common in plants, where extensive target
complementarity is the rule, and ancestral relationships between microRNAs (miRNAs)
and their targets can sometimes be detected; Drosophila hairpin RNA (hpRNA) may emerge similarly. MITE, miniature inverted-repeat transposable
element. Right: inverted repeats might also emerge from initially unstructured sequences.
This appears to be the dominant mode of miRNA emergence in animals. It also occurs
in plants, but only rarely do such miRNAs appear to acquire functional targets. (b) Inferred model for plant miRNA emergence from long foldbacks; arrows indicate evolutionary
relationships, arrowheads indicate small RNAs produced from a given hairpin. Long
hairpins are processed haphazardly, often by different Dicers, to generate heterogeneous
small interfering RNAs (siRNAs). As regulatory relationships are refined, the precision
and phasing of hairpin processing may increase. Shortening of the hairpin to produce
a single defined duplex may represent a mature state of plant miRNA evolution. (c) Expansion of miRNA clusters. In both plants and animals, local duplication may increase
the dosage of a given miRNA. In animals, there may be an advantage for Drosha cleavage
of hairpins emerging near extant miRNAs, leading to operons of unrelated miRNAs. (d) Different biogenesis mechanisms impose distinct demands on gene birth. Mirtrons need
only evolve the capacity for one RNase III cleavage by Dicer, whereas canonical miRNAs
need to gain the ability to be cleaved consecutively by Drosha and Dicer.

'Proto-miRNA' loci in plants are likely to transit through a stage where small RNAs
are imprecisely processed by one or more of the siRNA-generating DCL enzymes (Figure
2b). This hypothesis is supported by numerous examples of recently evolved plant MIRNA hairpins that are processed by DCL4, DCL3 or DCL2 instead of, or in addition to, the
canonical miRNA Dicer DCL1 [45,108-110]. In addition to generating multiple sizes of small RNAs (for example, 21, 22 and
24 nucleotides) characteristic of different Dicers, the small RNAs of a given size
may be only partially phased or unphased altogether. During this transitional period,
many small RNAs from the same foldback would be complementary to the target(s), thus
allowing beneficial regulatory relationships between the hairpin and the target to
be selected for without an immediate requirement for the precise processing that characterizes
canonical miRNAs. This functional, yet transitional, state may be suited to plants
(relative to animals) because of their requirement for a high degree of small RNA-target
complementarity, which may consequently minimize off-target effects.

A strategy for miRNA genesis from pre-existing RNA structures also seems to occur
with miniature inverted-repeat transposable elements (MITEs), whose eponymous inverted
repeats, when transcribed, create hairpin RNAs resembling proto-MIRNAs in plants [111]. A subset of animal miRNAs also derive from MITEs or other repetitive elements [112-114], and at least some of these may recognize mRNA targets bearing complementary repeat-related
sequences. However, as animal miRNAs rarely exhibit 'plant-like' extensive complementarity
to targets, the target duplication model does not seem to apply broadly in this kingdom.
It is worth considering the vertebrate mir-196 genes in this context. The three members of this family are located in homologous
positions within the four HOX genomic clusters, which encode conserved homeodomain
proteins that govern anterior-posterior identities of body segments. Several Hox genes
have miR-196 seed matches in their 3' UTRs, but the HOXB8 3' UTR has a highly conserved, fully complementary site to miR-196 [115,116]. The chromosomal proximity of mir-196a-1 to HOXB8 (separated only by HOXB9) is suggestively similar to the proximity of several young plant miRNA genes to their
targets, and raises the possibility that mir-196 genes evolved from a local duplication of HOX genes.

The Drosophila hpRNA pathway also offers an informative comparison. A number of endo-siRNAs generated
by hpRNA loci have clear targets bearing nearly perfectly complementary sites that
mediate their downregulation [64,65]. In the case of hp-CG18854 and its target CG8289, extensive target homology clearly indicates their ancestral relationship. This mimics
an early state in target-derived plant miRNA emergence. In the case of hp-CG4068,
the target mus308 bears a perfectly paired antisense target site to its most abundant endo-siRNA, but
lacks extended flanking homology. This may be analogous to a mature state in plant
miRNA history. We emphasize that there does not appear to be an evolutionary relationship
between hpRNAs and miRNAs in Drosophila (that is, there is no evidence that hpRNAs eventually become stabilized as miRNAs),
and the hpRNAs as a class are rapidly evolving. Nevertheless, the similarities between
Drosophila hpRNAs and plant miRNA genes are striking, apparently reflecting convergent evolutionary
strategies.

Emergence of miRNAs from initially unstructured sequences

Because vanishingly few animal miRNAs seem to have derived from their target genes,
it has long been assumed that a major route for miRNA birth in animals is through
de novo emergence of RNA hairpins that gain competence for miRNA biogenesis (Figure 2a). Recall that plant miRNAs commonly have one-to-one or one-to-a-few target relations,
but that animal miRNAs mediate broad regulatory networks owing to their minimal six-
to seven-nucleotide target pairing requirements. If we assume that gene regulation
in any extant individual is the product of substantial selective pressures for an
optimal state, the introduction of a novel regulatory RNA is likely to be either neutral
or detrimental, and only rarely beneficial [117,118]. In plants, the potentially detrimental influence of an emergent foldback on a miRNA
sequence target might be mitigated by increasing the activity or expression of that
target. However, in animals, one might imagine that emergent miRNA foldbacks might
have the potential to misregulate a large cohort of target genes, from which a return
to normalcy would not be easy.

Therefore, it has been posited that newborn miRNAs of animals are likely to 'creep'
quietly into existence, beginning with low expression levels whose regulatory activities
are tolerated by any targets encountered [117,118]. It also seems probable that de novo hairpins would not be fully endowed with characteristics permitting efficient miRNA
biogenesis, and this would also limit their maturation. Recent annotation efforts
in Drosophila melanogaster identified candidate hairpins that have evidence for miRNA biogenesis (that is, have
many reads, have star species, and/or have reads in Argonaute immunoprecipitates),
but do not show as clear evidence for precision of processing as do other more canonical
miRNA loci [52]. These include loci that clearly exhibit patterns of random RNA breakdown layered
on top of specific Drosha/Dicer-1/AGO1 biogenesis, suggesting that they are evolutionary
intermediates that are only partially processed by the miRNA pathway (Figure 3).

Figure 3.The complexity of annotating microRNAs from reads mapped to predicted hairpins. (a) Examples of loci that should not be annotated as microRNAs (miRNAs): hairpins with
single reads, heterogeneously sized reads, and/or putatively duplexed reads lacking
3' overhangs. (b) Examples of loci with some, but insufficient, evidence for miRNA biogenesis; such
loci are worth segregating as candidates pending further study. For instance, depletion
of reads from miRNA biogenesis mutants or enrichment in Ago complexes could elevate
their status from 'candidate' to 'confident'. It is also worth considering that candidate
miRNA hairpins with relatively imprecise processing patterns may represent transitional
intermediates in miRNA birth. nt, nucleotide. (c) Confident miRNA hairpins generate relatively precise miRNA/miRNA* duplexes with 3'
overhangs. As datasets grow, it is often possible to observe cloned terminal loops
or 5'/3' fragments (frag) of the primary miRNA (pri-miRNA) base, whose phasing with
miRNA/miRNA* termini provide stringent evidence for in vivo cleavage reactions. Note that the vertebrate locus mir-451 matures by direct hairpin cleavage by Ago2, and not via a miRNA/miRNA* intermediate;
thus, the criteria outlined in this figure are not applicable to mir-451-class substrates.

Under the appropriate circumstances, then, the occasional beneficial regulation mediated
by an emerging miRNA might be selected for. This would occur concomitantly with purging
of target sites that mediate detrimental regulation, otherwise favoring loss of the
emerging miRNA locus [117,118]. Such events might permit mutations within the hairpin that could improve its cleavage
by Drosha and/or Dicer enzymes, as well as improve transcriptional capacity. These
qualities are indeed mirrored in the general expression patterns of animal miRNAs.
Among highly expressed miRNAs, basically all are deeply conserved within a given clade
(for example, Drosophilids or vertebrates). The lowly expressed miRNAs include some
conserved loci, which might be due to their tissue-restricted expression, but essentially
all of the evolutionarily newborn miRNAs fall into the low-expression group [34,52,119].

Although the birth of many plant MIRNA loci can be explained by the target duplication model, comparisons between the closely
related species Arabidopsis thaliana and Arabidopsis lyrata have strongly implied that many MIRNA loci arose recently from inverted repeats formed from random intergenic sequences
[107,120,121]. Such MIRNA loci are likely to be rapidly lost due to mutational drift, as they are born without
pre-existing target homology; indeed, targets are not readily detectable for many
of these evolutionarily young miRNAs, and the patterns of nucleotide substitutions
in many of them suggest neutral drift rather than the constrained patterns of substitutions
found for older, clearly functional miRNAs [107,121]. Nevertheless, it has been inferred that targets can occasionally be captured for
young plant miRNAs. This is perhaps most clear in a few cases, where plant MIRNA hairpins born of genic duplications have acquired targets distinct from their originating
loci; for instance, Arabidopsis miR447 and miR856 have validated targets that are distinct from their loci of origin
[20,107].

Distinguishing bona fide miRNAs from RNA degradation products

The genomes of most higher eukaryotes are predicted to encode at least 105 to 106 putative hairpins with substantial similarity to validated pri-miRNA hairpins [122-124]. In theory, this constitutes an enormous reservoir of putative miRNA substrates,
whose trace regulatory activities might be subject to selection and evolutionary stabilization.
But how many predicted hairpins in a given genome are actually competent to be specifically
processed by the miRNA biogenesis machinery? Conserved miRNA genes are amenable to
computational discovery by signatures of hairpins that exhibit evolutionarily stable
arms and diverging terminal loops [122,123]. However, it is not currently possible to prospectively annotate the miRNAs encoded
by a genome, in the absence of comparative genomics, with any reasonable degree of
specificity or sensitivity. Therefore, the mere existence of large numbers of predicted
miRNA-like hairpins does not imply the existence of similar numbers of evolutionarily
emergent miRNA genes.

In light of broad transcription across euchromatin of plants and animals [125,126], including substantial amounts of transcribed sequence that are removed by quality
control mechanisms, it seems inevitable that short pieces of RNA will eventually be
associated with most of the genome, including most predicted genomic hairpins. For
example, many hundreds of candidate miRNA hairpins in various Drosophila species were initially annotated on the basis of singleton reads [119]. However, these were either not recovered in substantially larger datasets from the
same tissue sources, or generated heterogeneously sized reads mapping through the
predicted hairpins [127], indicating that few of these were genuine miRNAs produced by RNase III processing.

More recently, short RNAs were recovered from >100,000 hairpins in the D. melanogaster genome (from a starting set of nearly 1 billion short RNA reads from almost 200 libraries)
[52]. However, confident canonical miRNA production could only be assigned to approximately
200 loci. These included many miRNA hairpins that are recently or newly evolved in
D. melanogaster, but they did not include hundreds of other miRNA candidates predicted from comparative
analysis of the 12 sequenced Drosophilids [34,128]. Even though most of these predictions were associated with at least some small RNA
reads, none exhibited read patterns and/or sizes that were consistent with Drosha/Dicer-mediated
processing (Figure 3). Although caution should be exercised in interpreting negative evidence, such data
support the notion that a fairly limited number of genomic regions are competent for
recognition by the miRNA pathway when transcribed.

These findings are echoed by studies of other animals and plants. For example, while
novel mammalian miRNAs continued to be reported, a recent systematic analysis actually
revised the estimates of mammalian miRNAs downward, and called into question a substantial
subset of extant annotations [129]. Similarly in Arabidopsis, many hundreds of miRNA genes were predicted by comparing hairpins with inferred
target sites [130]. However, very few of these have since been validated by deep sequencing [20,45]. Altogether, these observations suggest that relatively few of the many predicted
genomic hairpins are substrates of miRNA biogenesis pathways, and hint that substantial
bioinformatic progress remains to bring miRNA prediction up to a par with protein-coding
gene prediction.

It is clear that more data on the efficiency and specificity of miRNA processing are
desirable, especially as all entries in the miRBase registry are currently treated
as equivalent [11]. Although some loci are now deemed suspect, in principle, the majority of entries
are confident miRNA genes that exhibit reasonably precise processing. The reality
is that with ever-increasing depths of small RNA sequencing, all miRNA loci exhibit
some level of terminal heterogeneity in their cloned products, and this tends to blur
the division between loci that can be confidently inferred to have transited an RNase
III pathway acting on a precursor hairpin, as opposed to fortuitous hairpins that
generate degradation products (Figure 3a). At present, a cautious approach to miRNA annotation seems warranted, in which miRNA
'candidates' are segregated from more confident miRNA loci [52]. These include loci that exhibit plausible miRNA/miRNA* duplexes but have substantial
reads not conforming to RNase III products, as well as hairpins lacking a star species
(Figure 3b). In particular, the lack of cloned miRNA* species was not previously seen as an
impediment to miRNA annotation; however, the depth of next-generation sequencing now
makes it reasonable to demand miRNA* species for confident annotation. (Exceptions
may be made with appropriate data; that is, if there is strong evidence from loss
of reads in miRNA biogenesis mutants, or enrichment of reads in Ago-IP samples.)

Perhaps more importantly, there are few data on how 'well' a miRNA hairpin is processed.
If only 1% of a miRNA hairpin transits the processing machinery, and these are processed
precisely, then it is equally eligible for annotation as loci for which 100% of the
hairpin is converted into miRNAs. The efficiency of processing cannot be inferred
from counts of small RNAs alone, since some endogenous miRNA loci might generate rare
reads owing to cell-specific expression, or perhaps post-transcriptional inhibition
of some aspect of their biogenesis [43]. It is only recently that efforts have been made to generate systematic data on the
effectiveness of miRNA processing, by cloning small RNA libraries from pools of cells
transfected with different miRNA expression constructs and quantifying the output
reads [129]. Larger scale data on forced expression of predicted hairpins, especially ones that
lack endogenous reads or exhibit heterogenous reads, should provide valuable insights
into the potentially partial capacity of some substrates to enter miRNA biogenesis
pathways.

Distinct evolutionary flux of different miRNA subclasses

In general, small RNAs that populate a miRNA-effector AGO have the functional attributes
of a miRNA, regardless of whether it was produced by a canonical or alternative pathway.
However, this does not mean that all miRNA substrates evolve similarly. For example,
the evolution of plant miRNAs from target duplications can be compared with those
from incidental hairpins. Because the plant miRNA system appears to require extensive
target complementarity, it is presumed that only rarely will fortuitous small RNA-generating
hairpins emerge and then subsequently acquire beneficial targets. Therefore, these
classes of plant miRNAs are expected to emerge and disappear with distinct dynamics
[106,107,120,121].

Does the genomic location of miRNA hairpins influence their emergence? Canonical miRNAs
in animals, unlike in plants, are commonly located in introns. Because animal miRNAs
mostly emerge from incidental foldbacks, the introns of bona fide transcription units might be a privileged location for miRNA birth already endowed
with directed primary transcription. We have also mentioned that animal miRNAs, unlike
their plant brethren, are frequently arranged in operons composed of dissimilar species.
This suggests that the emergence of animal miRNAs is privileged by location near an
extant miRNA (Figure 2c). Access to the Drosha/DGCR8 processing complex is the gatekeeper for entry into
the canonical miRNA pathway, and its activity has been reported to be co-transcriptional
[131-133]. Consequently, physical proximity of an emergent hairpin to an established miRNA
hairpin could enhance its nuclear cleavage relative to 'solo' emergent hairpins; this
might be particularly important for the processing of suboptimal hairpins. Newly evolved
miRNAs have indeed been detected in proximity to much more deeply conserved animal
miRNAs [52]. The lack of Drosha homologs in plants might render this characteristic less relevant
in the plant kingdom.

What is the influence of the biogenesis mechanism on miRNA evolution? If we compare
the canonical miRNAs with mirtrons (Figure 2d), a subset of mirtrons are well conserved in a given animal clade (that is, Drosophila or vertebrates), and these diverge more quickly in their terminal loop than their
hairpin arms [55,134], as seen with canonical miRNAs. However, relatively few mirtrons are well conserved
and no mirtrons are common between invertebrates and vertebrates, whereas many canonical
miRNAs are identical over this distance. This implied that mirtrons, as a class, emerge
and disappear more quickly than canonical miRNAs. This was confirmed with deep sequencing
of small RNAs from three Drosophilid species; this showed that mirtrons comprise a
steadily increasing fraction of confidently annotated miRNA loci as the evolutionary
branch length under consideration decreases [127]. Recent analysis of mirtrons from ultradeep sequencing has extended the species-specific
catalog of fly and nematode mirtrons further still [52,135].

The distinct evolution of mirtrons and canonical miRNAs might relate to the structural
hurdles needed to become a substrate of the respective biogenesis machineries (Figure
2d). In the case of canonical miRNAs, a substrate must simultaneously adopt conformations
that permit its cleavage by both Drosha and Dicer. In the case of mirtrons, a substrate
must be spliced and be a target of Dicer. We do not know how many endogenous substrates
of Drosha there are, but there are currently no more than some hundreds known in any
given animal species. By contrast, the many short constitutively spliced introns (for
example, there are 27,000 to 30,000 introns <120 nucleotides in flies and nematodes)
comprise a large pool of loci that have passed one processing hurdle. Structure-function
studies have shown that mirtrons are flexible with regard to primary sequence, provided
that they retain splicing functionality and adopt substantial hairpin structure with
3' overhangs [135]. In fact, the nature of splice sites (GU...YAG) should position many introns to pair
the 5' G with the 3' Y, leaving the AG as a two-nucleotide 3' overhang.

These factors might conspire to aid the evolutionary emergence of animal mirtrons,
relative to canonical miRNAs. Conversely, the apparent near absence of mirtrons in
plants could well be a consequence of a distinct biogenesis mechanism that does not
rely upon processing by two separate RNase III enzymes. Because plant miRNA maturation
relies upon a single Dicer-like protein to completely liberate a miRNA/miRNA* duplex
from a pri-miRNA transcript, the pre-miRNA intermediates produced by mirtrons may
be either unnecessary or perhaps even unrecognized by the plant DCL1 complex.

Concluding remarks

The major plant and animal miRNA pathways differ with respect to their biochemical
mechanisms, the extent of their preferred target pairing, and numbers of functional
targets. These differences have resulted in distinct characteristics of the evolution
of plant and animal miRNAs. In particular, the co-evolution of target:miRNA pairs
is common in plants, whereas it seems much more common for animal miRNAs to emerge
and then acquire target genes. One interpretation is that this reflects independent
emergence of miRNA pathways in plants and animals, on the backbone of an ancestral
RNAi pathway that metabolized dsRNA into short RNAs that populate Argonaute proteins.
This system might have emerged to defend against invasive nucleic acids such as viruses
and transposons, and subsequently been adapted to generate miRNAs from endogenous
inverted repeat transcripts. However, there are also analogies between plant and animal
miRNA pathways. For example, certain vertebrate miRNA targets, as well as Drosophila hpRNA targets, exhibit 'plant-like' extensive complementarity. There is reciprocally
a growing appreciation that plant miRNAs have emerged from incidentally emerged hairpins,
akin to the presumed dominant mode for animal miRNA birth. Therefore, an alternative
interpretation is that a miRNA pathway was extant in the last common ancestor of plants
and animals, but became differentially deployed in these kingdoms.

In either case, it is clear that a limited set of core proteins, namely RNase III
enzymes and Argonaute proteins, have been joined in remarkably diverse ways to control
gene expression via small RNAs. Recent studies of fungal small RNA pathways provide
additional evidence for innovation of RNase III-independent mechanisms for siRNA and
miRNA production [136,137], for which we can only guess at the underlying reasons that permitted the loss of
canonical pathways and invention of new pathways. Altogether, it is evident that miRNAs
are not a unitary entity, but instead encompass a variety of conceptually related
phenomena, whose evolutionary pressures differ according to mechanism of biogenesis
and even genomic location. Understanding the principles that govern the evolutionary
flux of these myriad small RNA pathways will provide a fundamental complement to understanding
the flux of protein-coding genes [138].

Acknowledgements

Work in MJA's group was supported by the NSF (MCB-0718051 and MCB-0964859), the NIH
(R01-GM084051), the Searle Scholar's Program, and the Arnold and Mabel Beckman Foundation.
JOW was supported by the Swedish Science Research Council. Work in ECL's group was
supported by the Burroughs Wellcome Fund, the Starr Cancer Consortium (I3-A139), and
the NIH (R01-GM083300 and U01-HG004261).